DC3, the 21-kDa Subunit of the Outer Dynein Arm-Docking Complex (ODA-DC), Is a Novel EF-Hand Protein Important for Assembly of Both the Outer Arm and the ODA-DC

The outer dynein arm-docking complex (ODA-DC) is a microtubule-associated structure that targets the outer dynein arm to its binding site on the flagellar axoneme ( Takada et al. 2002 . Mol. Biol. Cell 13, 1015–1029). The ODA-DC of Chlamydomonas contains three proteins, referred to as DC1, DC2, and DC3. We here report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 Da protein with four EF-hands that is a member of the CTER (calmodulin, troponin C, essential and regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming because of loss of some but not all outer dynein arms. Some outer doublet microtubules without arms had a “partial” docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly.

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EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK–calmodulin interaction

The Journal of General Physiology ◽

10.1085/jgp.200910295 ◽

2009 ◽

Vol 134 (4) ◽

pp. 281-293 ◽

Cited By ~ 44

Author(s):

Weiyan Li ◽

David B. Halling ◽

Amelia W. Hall ◽

Richard W. Aldrich

Keyword(s):

Modular Design ◽

Binding Capacity ◽

Current Model ◽

Sk Channels ◽

Channel Activity ◽

Wild Type ◽

Sk Channel ◽

Ef Hand ◽

Ef Hands ◽

Small Conductance

Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca2+ via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca2+ and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca2+-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca2+. These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca2+-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca2+ affinity than WT CaM.

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Hybrid myosin light chains containing a calcium-specific site from troponin C

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1992.tb16608.x ◽

1992 ◽

Vol 204 (1) ◽

pp. 85-91 ◽

Cited By ~ 3

Author(s):

Ana Claudia Rasera SILVA ◽

John KENDRICK-JONES ◽

Fernando C. REINACH

Keyword(s):

Troponin C ◽

Specific Site ◽

Light Chains ◽

Myosin Light Chains

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Tau interactome analyses in CRISPR-Cas9 engineered neuronal cells reveal ATPase-dependent binding of wild-type but not P301L Tau to non-muscle myosins

Scientific Reports ◽

10.1038/s41598-019-52543-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Xinzhu Wang ◽

Declan Williams ◽

Iris Müller ◽

Mackenzie Lemieux ◽

Ramona Dukart ◽

...

Keyword(s):

Protein Interactions ◽

Dendritic Spines ◽

Mitochondrial Fission ◽

Light Chains ◽

Myosin Light Chains ◽

Wild Type ◽

Quantitative Mass Spectrometry ◽

Steady State Levels ◽

P301l Mutation ◽

Frontotemporal Dementias

Abstract Protein interactions of Tau are of interest in efforts to decipher pathogenesis in Alzheimer’s disease, a subset of frontotemporal dementias, and other tauopathies. We CRISPR-Cas9 edited two human cell lines to generate broadly adaptable models for neurodegeneration research. We applied the system to inducibly express balanced levels of 3-repeat and 4-repeat wild-type or P301L mutant Tau. Following 12-h induction, quantitative mass spectrometry revealed the Parkinson’s disease-causing protein DJ-1 and non-muscle myosins as Tau interactors whose binding to Tau was profoundly influenced by the presence or absence of the P301L mutation. The presence of wild-type Tau stabilized non-muscle myosins at higher steady-state levels. Strikingly, in human differentiated co-cultures of neuronal and glial cells, the preferential interaction of non-muscle myosins to wild-type Tau depended on myosin ATPase activity. Consistently, transgenic P301L Tau mice exhibited reduced phosphorylation of regulatory myosin light chains known to activate this ATPase. The direct link of Tau to non-muscle myosins corroborates independently proposed roles of Tau in maintaining dendritic spines and mitochondrial fission biology, two subcellular niches affected early in tauopathies.

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Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.

The Journal of Cell Biology ◽

10.1083/jcb.119.6.1613 ◽

1992 ◽

Vol 119 (6) ◽

pp. 1613-1624 ◽

Cited By ~ 121

Author(s):

B E Taillon ◽

S A Adler ◽

J P Suhan ◽

J W Jarvik

Keyword(s):

Amino Acid ◽

Basal Body ◽

Calcium Binding ◽

Mutational Analysis ◽

Structural Defects ◽

Wild Type ◽

Immunofluorescence Analysis ◽

Ef Hand ◽

Level 1 ◽

Ef Hands

Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrin-containing fibrous structures associated with the Chlamydomonas centrosome.

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Homology of myosin light chains, troponin-C and parvalbumins deduced from comparison of their amino acid sequences

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(74)90927-9 ◽

1974 ◽

Vol 58 (1) ◽

pp. 301-308 ◽

Cited By ~ 106

Author(s):

John H. Collins

Keyword(s):

Amino Acid ◽

Troponin C ◽

Amino Acid Sequences ◽

Light Chains ◽

Myosin Light Chains

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Roles of individual EF-hands in the activation of m-calpain by calcium

Biochemical Journal ◽

10.1042/bj3480037 ◽

2000 ◽

Vol 348 (1) ◽

pp. 37-43 ◽

Cited By ~ 15

Author(s):

Previn DUTT ◽

J. C. Simon ARTHUR ◽

Pawel GROCHULSKI ◽

Miroslaw CYGLER ◽

John S. ELCE

Keyword(s):

Driving Force ◽

Specific Activity ◽

Small Subunit ◽

Wild Type ◽

X Ray ◽

X Ray Crystallography ◽

Kd Values ◽

Ef Hand ◽

Ef Hands ◽

Made In

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca2+-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca2+-binding EF-hands in each subunit, but their relative affinities for Ca2+ are not known. In the present study mutations were made in both subunits to reduce the Ca2+-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca2+ did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca2+ were otherwise identical to that of the Ca2+-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca2+ requirement (Kd) was approx. 350 μM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in Kd, which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain Kd values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca2+ and provides the major driving force for conformational change.

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Structural elements within the methylation loop (residues 112–117) and EF hands III and IV of calmodulin are required for Lys115 trimethylation

Biochemical Journal ◽

10.1042/bj3400417 ◽

1999 ◽

Vol 340 (2) ◽

pp. 417-424 ◽

Cited By ~ 2

Author(s):

Jennifer A. COBB ◽

Chang-Hoon HAN ◽

David M. WILLS ◽

Daniel M. ROBERTS

Keyword(s):

Single Point ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Structural Elements ◽

Nad Kinase ◽

Wild Type ◽

Loop Sequence ◽

Ef Hand ◽

Single Point Mutations ◽

Ef Hands

Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114 → Ala; L116T, Leu116 → Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.

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Sequential disassembly of myofibrils induced by myristate acetate in cultured myotubes.

The Journal of Cell Biology ◽

10.1083/jcb.105.3.1365 ◽

1987 ◽

Vol 105 (3) ◽

pp. 1365-1376 ◽

Cited By ~ 32

Author(s):

Z X Lin ◽

J R Eshelman ◽

S Forry-Schaudies ◽

S Duran ◽

J L Lessard ◽

...

Keyword(s):

Troponin C ◽

Stress Fibers ◽

Light Chains ◽

Myosin Light Chains ◽

Muscle Actin ◽

Myristate Acetate ◽

Thin Filaments ◽

Thick Filaments ◽

Cultured Myotubes ◽

Alpha Actin

The phorbol ester TPA induces the sequential disassembly of myofibrils. First the alpha-actin thin filaments are disrupted and then, hours later, the myosin heavy chain (MHC) thick filaments. TPA does not induce the disassembly of the beta- and gamma-actin thin filaments of stress fibers in presumptive myoblasts or fibroblasts, nor does it block the reemergence of stress fibers in 72-h myosacs that have been depleted of all myofibrillar molecules. There are differences in where, when, and how myofibrillar alpha-actin and MHC are degraded and eliminated from TPA-myosacs. Though the anisodiametric myotubes have begun to retract into isodiametric myosacs after 5 h in TPA, staining with anti-MHC reveals normal tandem A bands. In contrast, staining with mAb to muscle actin fails to reveal tandem I bands. Instead, both mAb to muscle actin and rhophalloidin brilliantly stain numerous disk-like bodies approximately 3.0 micron in diameter. These muscle actin bodies do not fuse with one another, nor do they costain with anti-MHC. All muscle actin bodies and/or molecules disappear in 36-h myosacs. The collapse of A bands is first initiated in 10-h myosacs. Their loss correlates with the appearance of immense, amorphous MHC patches. MHC patches range from a few micrometers to over 60 micron in size. They do not costain with antimuscle actin or rho-phalloidin. While diminishing in number and fluorescence intensity, MHC aggregates are present in 30% of the 72-h myosacs. Myosacs removed from TPA rapidly elongate, and after 48 h display normal newly assembled myofibrils. TPA reversibly blocks incorporation of [35S]methionine into myofibrillar alpha-actin, MHC, myosin light chains 1 and 2, the tropomyosins, and troponin C. It does not block the synthesis of beta- or gamma-actins, the nonmyofibrillar MHC or light chains, tubulin, vimentin, desmin, or most household molecules.

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